The traditional strategies for vaccine development have been to make killed, attenuated or subunit preparations as homologous prime/boosts, and then to test them for safety and efficacy1,2. Vaccines developed in this way are used world-wide for both bacterial and viral infectious diseases1-4. A number of viral targets have so far resisted this classical vaccine-development scheme—HIV-1, dengue and hepatitis C among them5-7. Broadly protective influenza vaccines have also yet to be been achieved8. HIV-1 is thus a paradigm of those viral diseases for which inducing broadly neutralizing antibodies is especially difficult9,10.
For many of the viral vaccines in current use, induction of neutralizing antibodies is a principal correlate of protection3, 4. Efforts to find new vaccine-development strategies have therefore focused on design of immunogens bearing epitopes with high affinity for plasma antibodies produced by memory B cells. This strategy assumes that the antigens recognized by memory B cells in a vaccine boost are the same as those recognized by naïve B cells during the priming immunization. For both HIV-1 and influenza, however, this strategy has not, as yet, led to induction in a majority of vaccines of antibodies that neutralize a satisfactorily wide range of virus strains. The failure may stem in part from characteristics of the chosen immunogens (e.g., glycan masking of HIV-1 envelope protein epitopes9: Table 1) and in part from limited accessibility of conserved epitopes on the viral antigen8 (e.g., the “stem” and sialic-acid binding epitopes on influenza HA). Mimicry of host antigens by some of these conserved epitopes may be another complication, leading to suppression of a potentially useful antibody response11.
TABLE 1Factors preventing induction of long-lasting broad neutralizing HIV-1 antibodiesNeutralizing epitopes masked by carbohydratesConformational flexibility of HIV-1 envelopeTransient neutralizing epitope expressionMolecular mimicry of Env carbohydrates and protein regions of hostmoleculesTolerance control of gp41 neutralizing epitope responsesHalf-life of all induced antibodies to Env are short; failure of Env to induce long-lived plasma cellsRapid viral escape from induced neutralizing antibodiesDiversion of B cell responses from neutralizing determinants by immune dominant, non-neutralizing epitopes of EnvRequirement for extensive somatic hypermutations, and requirement for complex maturation pathways
Making vaccines for infectious agents with transient, cryptic or host-mimicking epitopes may require detailed understanding of antibody affinity maturation—in particular, of patterns of maturation that lead to rare, broadly protective antibodies12-14. It might then be possible to design immunogens that increase the likelihood of maturation along those pathways. Recent data from animal studies have demonstrated that the B cells that survive and persist in the germinal center reaction are those presenting B-cell receptors with the highest affinity for antigen15-18. Moreover, for some responses to viral antigens, the antigen that stimulates memory B cells during affinity maturation and the antigen that initially elicits naïve B cells may not be the same12-14, 19-21. Thus, to induce the processes that lead to such a protective response, it may be necessary to use one antigen for the vaccine prime (to trigger naïve B cells) and others in boosts that drive affinity maturation12-14, 20-23.
Described herein is an approach to vaccine design based on insights from basic B cell biology, structural biology, and new methods for inferring unmutated ancestor antibodies as estimates of naïve B-cell receptors and their clonal lineage progeny. While the focus is on the biology of inducing broadly neutralizing antibodies to the HIV-1 Env, parallels are also drawn to issues for influenza vaccine development.
Biology of B Cells and Antibody Responses.
Human B cells arise from committed progenitors that express the V(D)J recombinase, RAG1 and RAG2, to effect genomic rearrangements of the IGH gene loci24-27. In pre-B I cells, functional μH polypeptides formed by these rearrangements associate with surrogate light chains (SLC)28-30 and Igα/Igβ heterodimers to form pre-B cell receptors (pre-BCR)31 necessary for cell survival and proliferation24, 32, 33. These cells exit the cell cycle25 as pre-B II cells, initiate rearrangements in the κ- or λ light (L)-chain loci34, 35, and assemble a mature BCR36, 37 that binds antigen24, 38 (FIG. 1). The generation of a BCR by genomic rearrangement and the combinatorial association of IG V, D, and J gene segments ensures a diverse primary repertoire of BCR and antibodies but also produces self-reactive cells with significant frequency39.
Most immature B cells are autoreactive; they are consequently eliminated or inactivated by immunological tolerance40, 41. The remaining B cells mature through the transitional 1 (T1) and T2 stages characterized by changes in membrane IgM (mIgM) density, mIgD expression, and the loss/diminution of CD10 and CD3842. In the periphery, newly formed (T2) B cells are subject to a second round of immune tolerization before entering the mature B cell pools40, 41. Each of these stages in B-cell development is defined by a characteristic genomic and physiologic status (FIG. 1); in concert, these events specify the potential of humoral immunity.
At least three mechanisms of immunological tolerance deplete the immature and maturing B-cell pools of self-reactivity: apoptotic deletion43, 44, cellular inactivation by anergy45, 46, and the replacement of autoreactive BCR by secondary V(D)J rearrangements39, 47-49. The great majority of lymphocytes that commit to the B-cell lineage do not reach the immature B cell stage because they express dysfunctional μH polypeptides and cannot form a pre-BCR50, 51 or because they carry self-reactive BCR40.
Autoreactive BCR frequencies decline with increasing developmental maturity43, 47, even for cells drawn from peripheral sites [FIG. 1]52, 53. The final stages of B-cell development and tolerization occur in secondary lymphoid tissues where newly formed (T2) B cells undergo selection into mature B-cell compartments54, 55. Tolerance mechanisms, especially apoptotic deletion54-56, operate during the transitional stages of B-cell development, and the frequency of self-reactive cells decreases substantially after entry into the mature pools40. The effects of these tolerizing processes have been followed directly in humans by recovering and expressing IgH and IgL gene rearrangements from individual immature, transitional, or mature B cells and determining the frequencies at which the reconstituted Abs react with human cell antigens40, 47.
Despite the multiple tolerance pathways and checkpoints, not all autoreactive B cells are removed during development41. In mice, mature follicular B cells are substantially purged of autoreactivity, but the marginal zone (MZ) and B1 B cell compartments are enriched for self-reactive cells57. In humans, some 20%-25% of mature, naïve B cells circulating in the blood continue to express autoreactive BCR35, 40, 41.
Not all selection during B-cell development is negative. Careful accounting of VH gene segment usage in immature and mature B-cell populations suggests that positive selection also occurs in the transitional stages of B-cell development58, 59, but the mechanisms for such selection are obscure. The substantial selection imposed on the primary B-cell repertoire, negative and positive, by these physiologic events implies that the full potential of the primary, or germline, BCR repertoire is not available to vaccine immunogens. Only those subsets of naïve mature B cells that have been vetted by tolerance or remain following endogenous selection can respond. For microbial pathogens and vaccine antigens that mimic self-antigen determinants, the pool of mature B cells capable of responding can, therefore, be quite small or absent altogether.
This censoring of the primary BCR repertoire by tolerance sets up a road block in the development of effective HIV-1 vaccines as the success of naïve B cells in humoral responses is largely determined by BCR affinity15-17. If immunological tolerance reduces the BCR affinity and the numbers of naïve B cells that recognize HIV-1 neutralizing epitopes, humoral responses to those determinants will be suppressed. Indeed, HIV-1 infection and experimental HIV-1 vaccines are very inefficient in selecting B cells that secrete high affinity, broadly neutralizing, HIV-1 antibodies5, 60-62.
The predicted effects of immune tolerance on HIV-1 BnAb production has been vividly illustrated in 2F5 VDJ “knock-in” (2F5 VDJ-KI) mice that contain the human VDJ gene rearrangement of the 2F5 BnAb61, 62. In 2F5 VDJ-KI mice, early B-cell development is normal, but the generation of immature B cells is severely impaired in a manner diagnostic of tolerization of auto-reactive BCR43, 44. Subsequent studies show that the 2F5 mAb avidly binds both mouse and human kynureninase, an enzyme of tryptophan metabolism, at an α-helical motif that matches exactly the 2F5 MPER epitope: ELDKWA63 (SEQ ID NO: 1) (Yang, G., Haynes, B. F., Kelsoe, G. et al., unpublished)
Despite removal of most autoreactive B cells by the central and peripheral tolerance checkpoints40, 41, antigen-driven, somatic hypermutation in mature, germinal center (GC) B cells generate de novo self-reactivity, and these B cell mutants can become memory B cells64-66. Thus, Ig hypermutation and selection in GC B cells not only drive affinity maturation15, 18, 67-69, but also create newly autoreactive B cells that appear to be controlled only weakly43, 70-72 by immunoregulation. At least two factors limit this de novo autoreactivity: the availability of T-cell help18, 73 and the restricted capacity of GC B cells to accumulate serial mutations that do not compromise antigen binding and competition for cell activation and survival18, 67, 74.
Eventually, V(D)J hypermutation approaches a ceiling, at which further mutation can only lower BCR affinity and decrease cell fitness73-75. The mean frequency of human Ig mutations in secondary immune responses is roughly 5%20, 76, 77, and the significantly higher frequencies (10%-15%) of mutations in Ig rearrangements that encode HIV-1 BnAbs5, 11 therefore suggest atypical pathways of clonal evolution and/or selection. In contrast to clonal debilitation by high mutational burden73-75, HIV-1 BnAbs appear to require extraordinary frequencies of V(D)J misincorporation5, 11. Perhaps the most plausible explanation for this unusual characteristic is serial induction of Ig hypermutation and selection by distinct antigens. This explanation also suggests pathways for generating antibody responses that are normally proscribed by the effects of tolerance on the primary BCR repertoire.
In GC, clonally related B cells rapidly divide; their clonal evolution is a Darwinian process comprising two component sub-processes: Ig hypermutation and affinity-dependent selection18, 67, 78. Selection is nonrandom of course, but even hypermutation is non-random, influenced substantially by local sequence context79 due to the sequence specificity of activation-induced cytidine deaminase (AICDA)80. Furthermore, the codon bias exhibited by Ig genes increases the likelihood of mutations in the regions that encode the antigen-binding domains81. Even prior to selection, therefore, some evolutionary trajectories are favored over others. Continued survival and proliferation of GC B cells is strongly correlated with BCR affinity and appears to be determined by each B cell's capacity to collect and present antigen18, 67 to local CXCR5+CD4+ T (TFH) cells82.
Unlike AICDA-driven hypermutation, where molecular biases remain constant, clonal selection in GC is relative to antibody fitness (affinity and specificity) and changes during the course affinity maturation. Individual GC, therefore, represent microcosms of Darwinian selection, and each is essentially an independent “experiment” in clonal evolution that is unique with regard to the founding B and T cell populations and the order and distribution of introduced mutations.
The poor efficiency with which either infection or immunization elicits BnAbs and the unusually high frequency of Ig mutations present in most BnAb gene rearrangements imply that BnAb B cells are products of disfavored and tortuous pathways of clonal evolution. Because BCR affinity is the critical determinant of GC B cell fitness, it should be possible to select a series of immunogens that direct GC B-cell evolution along normally disfavored pathways. Any method for directed somatic evolution must take into account the complex and interrelated processes of Ig hypermutation, affinity-driven selection, and cognate interaction with TFH. These hurdles are not insignificant, but neither are they necessarily insurmountable. Indeed, BnAb responses elicited by HIV-1 infection may represent an example of fortuitous sequential immunizations that, by chance, favor the development of BnAb B cells from unreactive, naïve populations.
Biology of Antibody Responses to HIV-1 as a Paradigm of Difficult-to-Induce Broadly Neutralizing Antibodies
The initial antibody response to HIV-1 following transmission is to non-neutralizing epitopes on gp4120, 83. This initial Env antibody response has no anti-HIV-1 effect, as indicated by its failure to select for virus escape mutants83. The first antibody response that can neutralize the transmitted/founder virus in vitro is to gp120, is of extremely limited breadth, and appears only ˜12-16 weeks after transmission84, 85.
Antibodies to HIV-1 envelope that neutralize a broad range of HIV-1 isolates have yet to be induced by vaccination and appear in only a minority of subjects with chronic HIV-1 infection5 (FIG. 2). Indeed, only ˜20% of chronically infected subjects eventually make high levels of broadly neutralizing antibodies, and then not until after ˜4 or more years of infection86. Moreover, when made, broadly neutralizing antibodies are of no clinical benefit, probably because they have no effect on the well-established, latent pool of infected CD4 T cells86.
Goals for an HIV-1 Vaccine
Passive infusion of broadly neutralizing human monoclonal antibodies (mAbs) can protect against subsequent challenge with simian-human immunodeficiency viruses (SHIVs) at antibody levels thought to be achievable by immunization87-90. Thus, despite the obstacles, a major goal of HIV-1 vaccine development is to find strategies for inducing antibodies with sufficient breadth to be practically useful at multiple global sites.
Recent advances in isolating human mAbs using single cell sorting of plasmablasts/plasma cells20,76 or of antigen-specific memory B cells decorated with fluorescently labeled antigen protein91, 92, and clonal cultures of memory B cells that yield sufficient antibody for high throughput functional screening22, 93, 94, have led to isolation of mAbs that recognize new targets for HIV-1 vaccine development (FIG. 2). Those broadly neutralizing antibodies that are made in the setting of chronic HIV-1 infection have one or more of the following unusual traits: restricted heavy-chain variable region (VH) usage, long HCDR3s, a high level of somatic mutations, and/or antibody polyreactivity for self or other non-HIV-1 antigens (rev. in5, 11). Some of these HIV BnAbs have been reverted to their unmutated ancestral state and found to bind poorly to native HIV-1 Env12, 14. This observation has suggested the notion of different or non-native immunogens for priming the Env response followed by other immunogens for boosting12-14, 20-23. Thus, the B cell lineage design strategy described herein is an effort to drive rare or complex B cell maturation pathways.